The present invention was made utilizing funds from contracts NO1-AI-72623 and NO1-AI-45179 between the National Institute for Allergy and Infectious Diseases and Georgetown University.
The present invention relates to compositions for the treatment of Hepatitis B virus (HBV) infection. In particular, the invention relates to antisense oligonucleotides and their use to inhibit HBV replication.
Hepatitis B virus, a member of the Hepadnaviridae family, is a blood-borne, hepatotropic pathogen which infects large numbers of people annually. In 1987 there were approximately 25,000 newly reported cases of disease attributable to HBV infection in the US. It is estimated that 60-70% of HBV infections lead to subclinical (asymptomatic) disease, and it is therefore probable that the actual number of new HBV infections each year is much higher. Of those infected with HBV, 90% make a full recovery, but 2-10% of infected patients develop chronic, persistent HBV infection. It is estimated that 1 million people in the US and 300 million people worldwide are chronically infected with HBV. In parts of Asia and Africa it is thought that between 5 and 20% of the population are chronically infected with HBV.
There is strong evidence linking chronic HBV infection to the incidence of primary hepatocellular carcinoma (HCC). Epidemiological studies demonstrate a much higher rate of HCC in regions of the world where HBV infection is endemic than in regions where infection is relatively low. A study in Taiwan demonstrated that HBV carriers (those chronically infected with the virus) were over two hundred times more likely to develop HCC as non-carriers. At the molecular level HBV DNA integration into the host genome has been demonstrated in nearly all hepatocellular carcinomas where HBV infection was present. This integration closely resembles that undergone by other tumor-causing viruses prior to malignant transformation. Over 500,000 deaths per year worldwide have been attributed to HCC, and hence a means of reducing the incidence of this cancer caused by HBV would be highly desirable.
Prior art has demonstrated that the course and outcome of HBV infections relates largely to the interaction of the duration and level of HBV replication and the degree of the host immune response. The severity of HBV-induced disease is, therefore, directly linked to HBV replication. See, for example, Purcell et al. "Hepatitis Viruses" In: DIAGNOSTIC PROCEDURES FOR VIRAL, RICKETTSIAL AND CHLAMYDIAL INFECTIONS. N. Schmidt et al., Ed. 6th Edition, pp 957-1065 (1989).
The only treatment for HBV currently licensed in the United States is alpha interferon. However this drug is not an effective treatment for all HBV chronic carriers and produces substantial side effects in some individuals during prolonged treatment. See, for example, Hoofnagle, "Current Status and Future Directions in the Treatment of Chronic Viral Hepatitis" In: VIRAL HEPATITIS AND LIVER DISEASE, Hollinger F. B. et al. (Eds.), pages 632-3. The majority of new approaches to the development of new antiviral agents against HBV have focused on the use of nucleoside analogues to inhibit the activity of viral polymerases. See Hoofnagle, supra. The limited success of this strategy against other viruses, together with the appearance of drug-resistant viral strains, indicates that this approach is unlikely to be a panacea.
A more recent approach to treatment of chronic HBV infection is the use of antisense oligonucleotides to inhibit viral gene expression. Goodarzi et al., J. Gen. Virol. 71: 3021 (1990); Wu et al., J. Biol. Chem. 267: 12436 (1992). The use of antisense oligonucleotides is known in the art. For a review, see Stein et al., Cancer Research 48: 2659 (1988). The method presents many technical hurdles however, and requires extensive experimentation if a successful outcome is to be achieved. See Stein and Chang, Science 261:1004 (1993). Use of the antisense method to inhibit replication of HBV requires knowledge of both the sequence of the HBV genome (to design suitable complementary oligonucleotides) and of the viral replication cycle (to target those regions of the genome critical to replication). Both the structure of the hepatitis B virus particle, known as a virion, and the viral replication cycle are well understood. See, for example, Tiollais et al., Nature 317:489 (1985), Wang and Seeger, J. Virol. 67:6507 (1993).
Goodarzi et al. prepared six antisense oligonucleotides against the S gene region of the HBV RNA pregenome, and measured their effects on HBsAg production in PLC/PRF/5 cells, which carry an unknown number of chromosomally integrated copies of HBV. Five of the six oligonucleotides proved effective at inhibiting production of HBsAg by the cells. The cell line used was not an ideal model for HBV replication in vivo however, since the HBV in this cell line is integrated as fragmented and rearranged pieces which cannot support HBV replication. Effects on HBV DNA and RNA were not measured.
Wu et al. described the use of a single antisense oligonucleotide targeted at the HBV polyadenylation signal/sequence, linked to a carrier intended to bind specifically to asialoglycoprotein receptors, which are abundant in the liver. The complex showed inhibition of HBsAg production, but the uncomplexed oligonucleotide was much less active. Again, effects on HBV DNA and RNA were not measured.
It is apparent therefore that new treatments for chronic HBV infection are greatly to be desired. In particular, it is greatly desirable to provide compositions and methods for treatment which are highly effective, but have a much lower incidence of side effects than those currently available.